Analogs of hirudin

ABSTRACT

A tyrosine-substituted hirudin analog has antithrombogenic activity. Further simultaneous reaction at native tyrosine residues is prevented by mutation at those sites to encode nonreactive amino acids. Several novel strategies for coupling the hirudin analog to solid surfaces while simultaneously conserving antithrombogenic activity are disclosed.

This is a continuation of application Ser. No. 08/116,939, filed on Sep.7, 1993, now abandoned which, is a continuation-in-part of U.S.application Ser. No. 07/747,565, filed Aug. 20, 1991, now abandoned.

TECHNICAL FIELD

This invention relates to analogs of hirudin and in particular relatesto, analogs of hirudin which have antithrombogenic activity and whichcan be bound to polymers.

BACKGROUND ART

Natural hirudin is a mixture of closely related polypeptides eachcontaining approximately 64 to 66 amino acids and having a molecularweight of approximately 6900 daltons. At least 20 natural variants ofhirudin have been identified. Scharf et al., FEBS Letters 255 pp. 105 to110 (September 1989). It is produced by the European medicinal leechHirudo medicinalis. It prevents blood from clotting by forming aninhibitory 1:1 molecular complex with activated thrombin (approximatelyK_(iApp=) 10⁻¹¹ to 10⁻¹⁴ M). Hirudin forms a very tight complex withthrombin, wherein over 40% of the hirudin structure intimately contactsthe thrombin molecule and covers both the fibrinogen recognition site ofthrombin and the fibrinogen cleaving (active) site of thrombin.Twenty-seven of the sixty-five residues of hirudin have contacts lessthan 4.0 Å with thrombin. This close fit prevents both the binding andcleavage of fibrinogen by thrombin.

It is impractical to prepare natural hirudin in quantities necessary fortherapeutic use. At least three recombinant hirudins are now availableidentical to native variants, except the recombinants lack the sulphateresidue on the tyrosine at position 63 found in native variants. Theserecombinant hirudins show pharmacological properties very similar tonative hirudin (Markwardt, Sem. Thromb. Hemostas. 15, pp. 269 to 282(1989)). European Patent Application 87402696.6 shows the amino acidsequence of hirudin variants 1, 2, and 3 (HV1, HV2 & HV3).

Three regions of the hirudin molecule are now believed to be essentialto the thrombin-hirudin high affinity interaction based on X-raycrystallography (Rydel, et al., Science 249 (1990) pp. 277 to 280) andstructure-activity studies (Krstenansky, et al., J. Med. Chem. 30 (1987)pp. 1688 to 1691). First, the three NH₂ -terminal amino acid residues atpositions 1, 2, and 3 of hirudin form a parallel beta strand with Ser²¹⁴to Glu²¹⁷ of thrombin and participate in several dozen non-polarinteractions with side chains of amino acids in and around the activesite of thrombin. Second, the NH₂ -terminal domain of hirudin fromapproximately Thr⁴ to Pro¹⁴⁸ is a compactly folded structure composed offour loops stabilized by three disulfide bridges and antiparallel betastructures. The main function of this domain is to position andfacilitate the interaction of hirudin's NH₂ -terminal tripeptide at thethrombin active site. Third, the COOH-terminal tail of hirudin (Glu⁴⁹-Pro⁶⁰) binds in the anion-binding exosite of thrombin and terminates ina hydrophobic helical turn defined by the sequence Glu⁶¹ -Leu⁶⁴. Theexosite constitutes the fibrinogen binding recognition site of thrombinand is dominated by numerous polar and non-polar interactions. Thepresence of hirudin in the exosite prevents fibrinogen from beingrecognized.

Most research on the hirudin molecule has focussed on (i) determiningthe roles of various regions of the molecule in its interaction withthrombin, and (ii) making modifications to the molecule to increase thebinding affinity between hirudin and thrombin and thereby reduce thenecessary dose in therapeutic applications. Some research has focussedon prolonging the activity or half-life of hirudin in vivo, and otherresearch has been in the area of immobilizing hirudin on surfaces usedin medical devices which come in contact with blood to provide anon-thrombogenic surface.

a. Modifications to Increase Binding Affinity or to Prolong In VivoHalf-Life of Hirudin

PCT Application WO 85/04418 discloses recombinant HV2 where Lys²⁴,Asn³³, Lys³⁵, Gly³⁶, Asn⁴⁷, Glu⁴⁹, and Asn⁵³ are replaced by Gln, Asp,Glu, Lys, Lys, Gln, and Asp respectively.

European Patent Application No. 87402696.6 discloses recombinantvariants 1, 2, and 3 where Tyr 63 or 64 is replaced by Glu or Asp andLys 47 or Asn 47 is replaced by Arg or His.

European Patent Application No. 89400621.2 also discloses amino acidsequence modifications, including those at positions 1, 2, 33, 35, 36,47, and 63, which increase the in vivo half life of the molecule,increase the specificity of the molecule's interaction with cell surfacereceptors and increase resistance to carboxypeptidase degradation. Argis placed in the 33 position, Thr or Ser or Asp are placed at position35, and Ser is placed at position 36.

European Patent Application No. 89810521.8 describes mutations at the53, 57, 58, 61, 62, and 63 positions which, depending upon the analogselected, provide increased or decreased antithrombogenic activity.

U.S. Pat. No. 4,179,337 discloses the attachment of mass-increasingmolecules such as polyethylene glycol to proteins.

Lazar, et al. describe mutations at position 3 in recombinant hirudinvariant 1 (rHV1) where antithrombin activity was increased by replacingTyr with Phe or Trp, and markedly decreased by replacement with Thr (J.B. Lazar, R. C. Winant & P. H. Johnson. J. Biol. Chem. 266 pp. 685-688(1991)).

Johnson, P. H. et al. in "Biochemistry and Genetic Engineering ofHirudin", Seminars in Thrombosis and Hemostasis, Vol. 15 No. 13 (1989)at pp. 309 describes hirudin fragments having antithrombogenic activity.These fragments correspond to residues 42 to 65 and 51 to 65.

European Patent Application No. 89810522.6 describes mutations atpositions 1, 27, 36, 47, 48, 49, 51 and 52. The substitution at 36 isLys, Arg, Asn, Val, Leu or Gln. The substitution at 27 is Gln, Asn, Leu,Arg, or Val. The substitution at 49 is Asn or Met. The substitution at47 is Arg, Asn, Val, or Leu.

European Patent Application No. 89810676.0 describes mutations atpositions 1, 2, 27, 36, 47, 57, 58, 61, and 66 where the position 27substitution is Gln, position 36 substitution is Gln and position 47substitution is Arg.

The following references disclose modifications at the C-terminal andN-terminal ends of the hirudin molecule: European Patent No. 142860;U.S. Pat. No. 4,801,576; U.S. Pat. No. 4,745,177; U.S. Pat. No.4,767,742, and European Application No. 86102462.8.

U.S. Pat. No. 4,791,100 discloses mutations of hirudin in positionscorresponding to, inter alia, 35 and 36, where at 35 the substitution isGlu and at 36 the substitution is Lys. It also discloses analogs havinga greater number of amino acids than native hirudin and others havingfewer amino acids than native hirudin.

b. Immobilization of Hirudin on Surfaces

European Application No. 89311022.1, European Application No. 89307922.8to Ito, and references cited therein disclose the attachment of hirudinto surfaces. The data disclosed in the Ito application shows substantialloss of antithrombogenic activity occurred when the molecule wasimmobilized on the surface.

c. Fragments

Various COOH-terminal polypeptide fragments of hirudin are known to bindto thrombin, thereby inhibiting the binding and cleavage of fibrinogenby thrombin. The minimum length polypeptide required to exert inhibitoryactivity has been reported as Phe ⁵⁶ -Gln⁶⁵ (J. L. Krstenansky, T. J.Owen, M. T. Yates & S. J. T. Mao. J. Med Chem 30 pp. 1688-1691 (1987)).Addition of amino acid residues to this polypeptide to increase itslength and provide the amino acid sequences found in the several hirudinvariants up to and including the complete sequences Glu⁴⁹ -Gln⁶⁵augments the thrombin inhibitory activity of the fragments; and, thesequence may be extended to include Gly⁴² -Gln⁶⁵ without compromisingthe efficacy of the inhibitor. Moreover, the deletion of Gln from thepolypeptides of these series provides an analogous series of usefulthrombin inhibitory peptides.

In a further extension of this reasoning, numerous synthetic variationsof the polypeptide sequences found in the natural hirudin variants(peptidomimetic analogs of hirudin peptides) have been prepared andfound to possess thrombin inhibitory activity. Notable among them arethose described in European Patent Application No. 89302159.2 and othersutilizing non-protein amino acids (European Patent Application No.89122451.1).

Maraganore, J. M. et al. in an abstract presented at a symposiumentitled "Biomedical Horizons of the Leech" on Oct. 24-28, 1990 disclosesynthetic peptides which bind to both the anion binding exosite and theactive site. The peptides are called "hirulogs" and consist of (i) anactive-site specificity sequence, (ii) a poly-Gly linker, and (iii) ananion binding exosite recognition sequence.

There are many variations possible on this model of bivalent thrombininhibitors and in general, bivalent protease inhibitors. For example seeJ. M. Maraganore, P. Bourdon, J. Jablonski, K. L. Ramachandran and J. W.Fenton, II. Biochemistry 29 pp. 7095-7101 (1990); J. DiMaio, B. Gibbs,D. Munn, J. Lefebvre, F. Ni, and Y. Konishi. J. Biol. Chem. 265 pp.21698-21703 (1990).

European Application No. 89302160.0 discloses peptides of about 8 to 26amino acids having the biological activity of hirudin.

European Application No. 89302159.2 discloses cyclicized syntheticfragments of hirudin having antithrombogenic activity.

It is a primary object of this invention to provide antithrombogenichirudin analogs having amino acids available for attachment of spacermolecules. The analogs can be bound to a surface via a spacer moleculerendering the surface nonthrombogenic. Alternatively, the analogs can bebound to a polymer via a spacer molecule to increase the analogs' invivo half life. It is a further object of this invention to providenonthrombogenic materials comprising such analogs attached to surfaces.It is a further object of this invention to provide analogs attached topolymers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid sequence of HV2-Phe³ Gln³³ Tyr³⁵ Lys⁴⁷ Asp⁶³and its encoding DNA sequence (SEQ ID NOS: 1&2).

FIG. 2 depicts the expression vector for the sequence in FIG. 1.

SUMMARY OF THE INVENTION

The present invention provides an analog of hirudin having at least oneamino acid in positions 30 to 37 substituted with Tyr, and the nativeTyr³ and Tyr^(63or64) residues substituted with a first and a secondfunctional nonreactive amino acid.

A preferred embodiment of the invention is an analog of hirudin havingat least one amino acid in positions 32 to 36 substituted with Tyr, thenative Tyr³ substituted with Phe, Ile or Leu, and Tyr^(63or64)substituted with Asp or Glu.

As used herein the term analog includes fragments and analogs of hirudinwherein a tyrosine residue is attached to the NH₂ -terminal position ofsuch analogs, and the residue equivalent to Tyr⁶³, when present, issubstituted with either Glu or Asp.

As used herein the term analog includes peptidomimetic analogs ofhirudin which are bivalent inhibitors of thrombin, where a tyrosineresidue is inserted in or near the oligomer that links the COOH-terminalhirudin peptide mimic to the peptide that binds in the active site ofthrombin, and where a functional nonreactive amino acid, preferably Aspor Glu, replaces the residue equivalent to Tyr⁶³.

The present invention also overcomes the disadvantages of the prior artby providing an antithrombogenic hirudin analog covalently attached to aspacer molecule at a reactive nonfunctional Tyr residue of the analog.

The present invention further provides Applicant's novel analogsattached to surfaces rendering such surfaces nonthrombogenic.

The present invention further provides Applicant's novel analogsattached to mass-increasing molecules, which will have a prolongedhalf-life in vivo.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides analogs of hirudin having at least one"reactive" amino acid in positions 30 to 37 and having a "functional"but "nonreactive" amino acid at positions 3 and 63. A prominent loop orfinger region extends out away from the hirudin-thrombin interface andcontains at its tip the sequence Leu³⁰ Gly³¹ Ser³² Asn³³ Gly³⁴ Lys³⁵Gly³⁶ Asn³⁷ (for hirudin variant 2). Amino acid substitutions in thisregion are believed not to affect the interaction of hirudin withthrombin. See European Patent Application No. 89400621.2 and Rydel, etal., Science 249 pp. 277 to 280 (1991). In accordance with the presentinvention this loop is a preferred site for substitution with Tyr toallow the attachment of a spacer molecule for surface immobilization ofa hirudin analog to render the surface nonthrombogenic. Alternately, inaccordance with the present invention, the novel hirudin analog may bebound to an oligomer, a polymer, a macromolecule, or othermass-increasing molecule, thereby increasing the effective molecularweight of hirudin and prolonging its in vivo half-life and itsanticoagulative effect in the circulation when administeredtherapeutically.

To avoid reaction of the spacer molecules or mass-increasing moleculewith Tyr in positions outside the finger region, where attachment to amolecule might destroy antithrombogenic activity, Tyr residues outsidethe finger region are substituted with amino acids that will not reactwith the spacer. As used herein, the term "nonreactive" shall mean anamino acid which, due to its nature and/or position within the analog,will not form a covalent bond with certain mass-increasing and spacermolecules described below. As used herein the term "functional" shallmean an amino acid in a particular position necessary for the analog tohave antithrombogenic activity.

Hirudin includes a Tyr at positions 3 and 63 or 64, which are functionalin the sense that they are believed to be necessary for the molecule tohave antithrombogenic activity (See European Patent Application No.87402696.6 and Lazar et al., op. cit.). However, the native Tyr at 3 and63 are also reactive. If not substituted these amino acids will reactwith the spacer or mass-increasing molecule rendering the productineffective as an antithrombogenic agent. European Patent ApplicationNos. 87402696.6 and 89810521.8 suggest that the substitution of Asp orGlu for the native Tyr at position 63 will not destroy theantithrombogenic activity of the analog. X-ray crystallographic studiesshowed that in a hirudin-thrombin complex, Tyr³ of hirudin occupies ahydrophobic cleft adjacent to the active site of thrombin that isoccupied in a similar manner by the Phe residue of the thrombininhibitor, PPACK (Phe-Pro-Arg-chloromethylketone) (Rydel, et al., op.cit.) This suggests that substitution of Phe for Tyr³ would notsignificantly reduce the antithrombogenic activity of the analog.

Accordingly, in the analog of the present invention, the functional Tyr³and Tyr⁶³ are replaced with functional yet nonreactive amino acids toprevent reaction of the spacer or mass-increasing molecule at thefunctional positions. The preferred functional, nonreactive amino acidsfor position 3 in hirudin are Phe, Leu and Ile. The preferredfunctional, nonreactive amino acids for position 63 or 64 are Asp andGlu. When both the finger region substitution and these terminalsubstitutions are made the result is an analog having Tyr available forreaction with a spacer or mass-increasing molecule in the nonfunctionalfinger region and functional, yet nonreactive amino acids in positions 3and 63 or 64.

In a preferred embodiment Tyr is placed at position 35 in hirudin. Tyrmay also preferably be placed at position 33.

As disclosed in European Application No. 87402696.6, in hirudin variant2 native Asn at 47 may be substituted with Lys to enhance the bindingaffinity of the molecule to thrombin.

The present invention requires at least one nonfunctional amino acidavailable for reaction. The inclusion of additional nonfunctionalreactive Tyr substitutions may enhance the usefulness of the analog forchemical attachment to surfaces or in promoting increased in vivo halflife of the analog. Those skilled in the art using routineexperimentation will be able to determine whether the introduction oftoo many of the disclosed Tyr substitutions in the analog will impairits usefulness in chemical attachment to surfaces or to mass-increasingmacromolecules, due to, for example, steric hindrance of the portions ofthe analog which bind to thrombin.

The analogs of the present invention may be prepared using recombinantDNA techniques known to those skilled in the art, for example, bysubjecting the gene that codes for hirudin to site-specific mutagenesisand expressing the mutated gene in a suitable host such as a yeast orbacterium. European Patent No. 200655 discloses an expression system forhirudin in yeast. The plasmid used to transform the yeast may be alteredby methods known to those skilled in the art to create the novelmutations described herein. European Patent Application Nos. 89810521.8and 89810522.6 of Ciba Geigy AG and patent applications cited thereindisclose microbial hosts for vectors containing hirudin DNA sequences.The analog described in Example 1 below was made by the methodsdisclosed in European Patent Applications Nos. 87401649.6 and89400621.2.

Those skilled in the art will understand that fragments of the hirudinmolecule, COOH-terminal polypeptide fragments, peptidomimetic analogs,and bivalent inhibitors (as described above in the section entitled"Fragments") may also be modified according to the criteria of thepresent invention provided that such "fragment" has at least one sitewhere a Tyr may be substituted without eliminating the antithrombinactivity of the "fragment", and further provided that the functionalgroups on the "fragment" are either nonreactive or can be substitutedwith a functional nonreactive amino acid. Fragments or peptides havingsuch activity and modifications thereof fall within the intent and scopeof the present invention. Accordingly, the term analog as used hereinshall include a fragment of the hirudin molecule, peptidomimeticanalogs, and bivalent inhibitors having antithrombogenic activity. Theterm analog as used herein shall include a synthetic peptide havingantithrombogenic activity by virtue of an amino acid sequence analogousto that of the functional portions of the native hirudin molecule.

For example, the NH₂ -terminal end of the hirudin peptides andpeptidomimetic analogs known to inhibit thrombin activity has a lesserinfluence on their effectiveness than the COOH-terminal residues. SeeJohnson, P. H. et al. in "Biochemistry and Genetic Engineering ofHirudin", Seminars in Thrombosis and Hemostasis, Volume 15, No 13 (1989)and J. L. Krstenansky, T. J. Owen, M. T. Yates, and S. J. T. Mao, J.Med. Chem. 30, PP. 1688-1691 (1987). Attachment of a Tyr residue to theNH₂ -terminal end or equivalent region of these various inhibitorypolypeptides, and peptidomimetic analogs, accompanied by substitution ofthe Tyr equivalent residue by Asp or Glu, provides a unique attachmentsite for an immobilizing spacer molecule or mass-increasing moleculeaccording to the methods of this invention.

The bivalent thrombin inhibitors described by Maraganore et al.,Biochem. 29, pp. 7095 to 7101 (1990) offer design flexibility in theplacement of a reactive amino acid residue for attachment of spacer ormass increasing molecules. A tyrosine residue inserted in or near theoligoglycine connecting link that joins the active site binding moietywith the longer peptide that binds in the fibrinogen recognition siteprovides a unique site for attaching a spacer, when in accordance withthis invention, the Tyr⁶³ equivalent residue is replaced with Glu orAsp.

Tyr is used for spacer attachment because it provides for site specificchemical reactions that avoid binding the spacer to other residues thatcould interfere with hirudin's activity. The specificity of thesereactions depends on the altered reactivity of groups inserted into thephenolic ring of Tyr. A preferred means to attach a spacer to a Tyrresidue is to insert a primary amine into its phenolic ring. Manyreagents developed for derivatization and immobilization of proteins aredesigned to react with primary amino groups in their neutral,unprotonated state. Use of these reagents with hirudin under usualderivatization conditions will impair the antithrombin activity ofhirudin by attaching spacer molecules to its NH₂ -terminal amine or tocertain of its lysyl epsilon-amino groups. An aryl amine on Tyr avoidsthese drawbacks by reacting with amine reactive agents under conditionsthat virtually exclude reactivity with the alkyl amines of Lys and theNH₂ -terminal amino acid residue. An aryl amine of Tyr has a pK_(a) ofabout 4.8, i.e., it is 50% protonated at pH 4.8. Since the protonatedform of a primary amine is unreactive to the commonly used spacerattachment chemistries, the tyrosyl amine residue will react at about50% efficiency at pH 5.0. However, at pH 5.0, less than 0.1% of thealpha-amine of the NH₂ -terminal residue (pK_(a) about 8.0), and lessthan 0.001% of the epsilon-amine of Lys (pK_(a) about 10.0) will bereactive with such spacer chemistries. This provides thesite-specificity. However, chemical methods that insert an amine intothe phenolic ring of Tyr may also react with Trp and free Cys; however,hirudin has no Trp residues and all 6 Cys residues are engaged inunreactive disulfide bridges. Therefore, specific reactivity with Tyr isobtained.

A less preferred means to attach spacers to Tyr uses spacers activatedwith diazonium salts, which react directly and efficiently with thephenolic ring of Tyr. However, this reaction is not specific to Tyr,since His residues also react and hirudin's only His is essential tothrombin binding. Other less preferred spacer chemistries used to attachspacers to Tyr residues are photo-oxidation, N-bromosuccinimide andsulfonyl halides which also react with amino acid side chains other thanTyr.

Spacers capable of reacting predominantly with aryl amines rather thanwith alkyl amines at about pH 5.0 include, but are not limited to, thosecontaining N-hydroxysuccinimidyl esters, imidate esters, thiolactones,carboxyanhydrides, sulfonyl halides, isourea esters, benzoquinones,vinyl sulfones, hydrazides and imidazolyl carbonyls. Typically suchspacer molecules are bifunctional, wherein one end of the spacercontains an amine-reactive chemical moiety, while the other end containsthe same or a different reactive species for attachment to the surface.

Whereas the spacer molecule is covalently bound to hirudin or itsanalogs in accordance with the present invention, the attachment of thespacer to the surface may occur by any binding means or combination ofbinding means, that will retain a sufficient concentration of hirudin orits analogs at the surface to provide a nonthrombogenic andanticoagulant surface under the conditions of use.

Attachment of the hirudin analog and its spacer to the surface may be bycovalent means, reacting the group on the free end of the spacer with areactive group on the surface. Alternately the hirudin-spacer conjugatemay be coupled to a reactive group on the free end of a different spaceror on a macromolecule which are themselves covalently bound to thesurface. The preferred chemical reactions to attach hirudin analogs to asurface or to a surface-bound spacer are those that occur rapidly andquantitatively under moderate conditions and avoid reaction withreactive amino acid side chains of hirudin and its analogs ordenaturation of the molecule. Examples of such reactive pairs include,but are not limited to, thiol-maleimide, thiol-iodoacetate, andhydrazide-aldehyde (from oxidized sugar cis-diols). Alternately, thehirudin analog and its spacer may be attached to the surface bynon-covalent binding means, which may include, for example, those thatoperate predominantly by hydrophobic binding mechanisms, or byfluorophilic associations, or by high affinity ligand receptor binding.The spacer attached to the hirudin analog may have at its free end, forexample, a hydrophobic or a fluorophilic moiety that will bind directlyto a similarly hydrophobic or fluorophilic surface. Or, the said spacermay have at its free end a chemical moiety that reacts to produce acovalent bond with the free end of a second spacer that is attached tothe surface by non-covalent means. In another embodiment, thehirudin-attached spacer may terminate in a high affinity ligand, such asa biotin molecule, which would then bind to its high affinity receptormolecule, such as avidin, that is itself covalently bound to thesurface. Or, where the high affinity receptor molecule has multiplebinding sites for its ligand, the receptor molecule may be attached tothe surface by binding to one of its specific ligands that is itselfattached to the surface by any of the covalent or non-covalent bindingmeans or combination of binding means just described.

The hirudin of this invention may be attached to materials which areuseful in the production and use of medical products, systems anddevices. Such materials include naturally occurring, genetically derivedand synthetic materials. Naturally occurring materials include tissues,membranes, organs and naturally occurring polymers. One example of agenetically derived material is poly-beta-hydroxybutyrate.

Such naturally occurring, genetically derived and synthetic polymershomo- and co-polymers derived from one or more of the following:1-olefins, such as ethylene, propylene, tetrafluoroethylene,hexafluoropropylene, vinylidene difluoride, etc.; vinyl monomers, suchas vinyl chloride, styrene, maleic anhydride, methylmethacrylate,acrylonitrile, etc.; ethers, such as ethylene, tetramethylene, etc.;esters, such as ethylene-terephthalate, bisphenol A-terephthalate, etc.;carbonates, such as bisphenol A, 4,4-dihydroxybiphenylene, etc.; amides(including ureas and urethanes), such as nylons, segmentedpolyurethanes, proteins, etc.; saccharides, such as glucose,glucosamine, guluronic acid, sulfated glycoseaminoglycans, agarose,alginic acid, etc.; siloxanes, such as dimethyl siloxane, 3-aminopropylsiloxane, etc. Polymers which are useful in this invention may includebiodegradable, partially biodegradable and non-biodegradable polymers.Other useful materials include metals, such as aluminum and stainlesssteel; glass, ceramics, and carbon in its various forms.

The choice of the material to which hirudin or its analogs may beattached generally depends on the function of the medical device orproduct incorporating that material. Given a specific material orcombination of materials in a single device, or system of multipledevices, a surface attachment strategy is formulated for hirudin,following principles and logic well known to those skilled in the art.The above considerations ultimately determine the chemical groupselected for the free end of the spacer attached to hirudin, and thesubsequent members of the chain that retains hirudin at the materialsurface.

It is understood that the mechanisms described for attachment of hirudinand its analogs to surfaces in accordance with the present invention areequally applicable to their, attachment to mass-increasing molecules forthe purpose of prolonging their in vivo half-life. Examples of suchmass-increasing molecules include, but are not limited to, polymers suchas polyethylene glycol or oxide, polyvinylpyrrolidone or thepolyglucoses; and macromolecules such as serum albumin, avidin, heparin,or hydroxyethyl starch. Large, globular mass-increasing molecules may beattached to hirudin by means of a long spacer that provides hirudin withsufficient spatial freedom to achieve its inhibitory position onthrombin; or, in other words, steric interference between themass-increasing molecule and thrombin must not block the presentation ofhirudin to its binding sites on thrombin. Polyethylene glycol or oxidechains, which are generally attached directly to the macromolecule ofinterest, demonstrate a mass-increasing effect beyond their actual massbecause of the larger excluded volume subtended by their highly mobilechains. See: Knauf, M. J. et al., J. Biol. Chem. 263 pp. 15064 to 15070(1988). Site-directed PEGylation of hirudin at the finger region Tyrpositions the mobile polyethylene glycol/oxide chains on the side of thehirudin molecule opposite from its thrombin-binding site.

EXAMPLE 1

Production of rHV2-Phe³ GLn³³ Tyr³⁵ LYS⁴⁷ Asp⁶³ (SEQ ID NOS: 1&2) HV2has Tyr at positions 3 and 63. In accordance with the present invention,the reactive functional Tyr³ is replaced with nonreactive functionalPhe, and the reactive functional Tyr⁶³ is replaced with nonreactivefunctional Asp. These replacements do not reduce the thrombin bindingactivity of the molecule. Reactive nonfunctional Tyr is then substitutedfor the native nonreactive nonfunctional Lys at position 35 in thefinger region. This change also does not eliminate the thrombin bindingactivity of the molecule but it does provide a site where Tyr isavailable for reaction. Asn at position 47 may be changed to Lys asdescribed in European Patent Application No. 87402696.6 to improve theactivity of the antithrombogenic analog.

The hirudin analog HV2 Phe³ GLn³³ Tyr³⁵ LYS⁴⁷ Asp⁶³ (SEQ ID NOS: 1&2)was prepared by the following methods:

A. Starting material

Starting material was phage M13TG4892. This phage (a derivative ofM13TG131) contains an expression block consisting of:

a. a slightly modified version of the MFαl promoter (the 5' EcoRI sitewas converted to a SphI site, and the internal BglII site was destroyedby a treatment with the Klenow fragment of E. coli DNA polymerase I),

b. a variant of the yeast BGL2 signal peptide (BGL2-Val⁷), and

c. the rHV2-Lys⁴⁷ coding sequence.

B. rHV2-Lys⁴⁷ Asp⁶³

By site directed mutagenesis (oligonucleotide OTG2942; Amersham sitedirected mutagenesis kit #RNP 1523) on single stranded DNA (ssDNA) ofM13TG4892 the codon TAT (Tyr⁶³) was exchanged for GAC (Asp) resulting inM13TG5884. The mutation was verified by sequencing the entire hirudincoding sequence (sequencing primer: OTG2387). rHV2-Lys⁴⁷ Asp⁶³ isencoded in M13TG5884.

C. rHV2-Phe³ Gln³³ Tyr³⁵ Lys⁴⁷ Asp⁶³

By site directed mutagenesis (oligonucleotides OTG2993: Tyr³ to Phe andOTG2994: Asn³³ to Gln, Lys³⁵ to Tyr) on ssDNA of M13TG5884 the codonsTAT (Tyr³) were exchanged for TTC (Phe), AAT (Asn ³³) for CAA (Gln), andAAG (Lys ³⁵) for TAC (Tyr) at the same time resulting in M13TC6844. Themutations were verified by sequencing the entire hirudin coding sequence(FIG. 1). rHV2-Phe³ GLn³³ Tyr³⁵ LYS⁴⁷ Asp⁶³ is encoded in M13TG6844.

D. Assembly of the expression vector pTG6864

The yeast basic expression vector pTG3828 (pBR322, 2 micron, URA3-d,PGK1 transcriptional terminator) was used to assemble the expressionplasmid. Vector pTG3828 and M13TG6844 (dsDNA) were digested with SphIand SalI and ligated. The ligation mixture was used to transform E. colistrain BJ5183 to ampicillin resistance (Ap^(R)). Plasmid DNA wasisolated from six Ap^(R) clones, and the PstI restriction profile ofeach preparation analyzed. Corresponding to the expected restrictionprofile clone N°1 was used for a CsCl purification of pTG6864 (alkalinelysis protocol). Structure of the purified plasmid was verified again bydigestion with PstI and SphI+Sal1.

pTG6864: the yeast rHV2-Phe³ GLn³³ Tyr³⁵ LYS⁴⁷ Asp⁶³ production plasmid(FIG. 2) is an E. coli-yeast shuttle vector with the following elements:

i. a bacterial segment, which is derived from E. coli plasmid pBR322,harboring a bacterial origin of replication (ori), and the bacterialselection marker for ampicillin resistance (Ap^(R))

ii. a segment of the yeast 2 micron episome with its origin ofreplication

iii. a promoter- and terminator-deleted version of the yeast URA3 gene(URA3-d) serving as a yeast selectable marker

iv. a modified version of the yeast MFα1 promoter

v. a sequence coding for a variant form of the yeast BGL2 derived signalpeptide serving as a secretion signal fused in frame to

the rHV2-Phe3 Gln33 Tyr³⁵ Lys47 Asp63 coding sequence, and

segment of the yeast PGK gene serving as a transcriptional terminator.

Thus, pTG6864 confers ampicillin resistance to transformed E. colicells; and it renders transformed yeast ura3 auxotrophic strainsprototrophic for uracil (Ura⁺).

The DNA sequence encoding rHV2-Phe³ Gln³³ Tyr³⁵ Lys⁴⁷ Asp⁶³ (FIG. 1) hasbeen verified after site directed mutagenesis, and is contained inM13TG6844 and pTG6864, respectively.

E. Transformation of yeast strain TGY48.1

Plasmid pTG6864 has been used to transform Saccharomvces cerevisiaestrain TGY48.1 MATαura3his3 pral prbl prcl cpsl to uracil prototrophy(lithium acetate protocol; 5.5 ,μg of plasmid DNA per 1.3×10⁸ cells).TGY48.1 is a haploid strain of mating type α (MATα) with a nonrevertingallele of the URA3 gene (ura3-Δ5) as selectable marker. After four daysincubation at 30° C. three Ura⁺ clones were obtained. Clone No°1 wasfurther analyzed.

F. Hirudin production from TGY48.1/pTG6864

Ura⁺ prototrophy of clone N°1 was verified. Clone No°1 was grown at 30°C. in an Erlenmeyer flask (250 ml) under selective conditions for 48 hrsto a final cell density (measured as absorbance at 600 nm, where A₆₀₀ =1corresponds to 10⁷ cells/ml) of 10 to 12. Cells were centrifuged out,and culture supernatants were tested for thrombin inhibition in akinetic assay using the chromogenic substrate,Tos-Gly-Pro-Arg-4-nitroanilide acetate (Chromozym TH, BoehringerMannheim, Germany). Hirudin production was expressed as theanti-thrombin activity of yeast culture supernatant (ATU/ml) normalizedto the A₆₀₀ of the culture.

EXAMPLE 2

Once an analog is prepared having a nonfunctional Tyr available, knownmethods may be employed to bind that Tyr to a spacer.

Hirudin containing an aryl amine on tyrosine (Hirudin-Tyr-NH₂) wasprepared by nitration followed by reduction (J. F. Riordan and B. L.Vallee. Methods Enzymol. 25 pp. 515-521 (1972)). The nitration reactionwas performed at room temperature in 0.01M sodium phosphate, pH 8.3, bymixing hirudin (0.7×10⁻⁶ M) with a ten-fold molar excess of ethanolictetranitromethane. The reaction was monitored by absorbance at 428nanometers for 2 hours, then terminated by gel filtration on BIO-GEL ®(cross-linked polycrylamide) P-6DG pre-equilibrated with the sodiumphosphate buffer. Hirudin-Tyr-NO₂ was reduced to hirudin-Tyr-NH₂ byadding a ten-fold molar excess of sodium dithionite in the sodiumphosphate buffer and incubating until the nitrophenol absorbance at 428nanometers disappeared. The aryl amino (tyrosine) hirudin was separatedfrom excess sodium dithionite by desalting on BIO-GEL ® P-6DGpre-equilibrated with 0.04M sodium acetate, pH 5.0, concentrated andstored at -20° C.

EXAMPLE 3

Attachment of SPDP to Hirudin-Tyr-NH₂

Sulfo-LC-SPDP (Sulfosuccinimidyl 6- 3-(2-pyridyldithio) propionamido!hexanoate) was attached to hirudin containing an aryl amine on tyrosineby the following method: To hirudin-Tyr-NH₂ (0.143×10⁻⁶ M) in 0.04Msodium acetate, pH 5.0, was added a ten-fold molar excess ofSulfo-LC-SPDP and the solution agitated for 2 hours at room temperature.Excess Sulfo-LC-SPDP was removed by desalting on BIO-GEL® P-6DGpre-equilibrated with 0.01M sodium phosphate, pH 7.0, and theSPDP-hirudin concentrated and stored at -20° C. The LC-SPDP-hirudin wasincubated before use with a 3-fold molar excess of dithiothreitol in0.04M sodium acetate, 0.005M EDTA, pH 4.5, until absorbance at 343nanometers reached a plateau, then the LC-SPDP-hirudin was purified bydesalting on BIO-GEL® P-6DG.

EXAMPLE 4

Binding N-Acetyl-homocysteine to Hirudin-Tyr-NH₂

N-Acetyl-homocysteine was attached to hirudin that contained an arylamine on tyrosine by the following method: Into a solution ofhirudin-Tyr-NH₂ (0.143×10⁻⁶ M) in 0.04M sodium acetate, pH 5.0, wasmixed a ten-fold molar excess of N-acetyl-homocysteine thiolactone(AHTL) in methanol and reaction continued for two hours with constantagitation at room temperature. Excess AHTL was removed by desalting onBIO-GEL® P-6DG pre-equilibrated with 0.01M sodium phosphate, pH 7.0, andthe N-acetyl-homocysteine-hirudin concentrated and stored at -20° C. Allreactions were performed under a stream of nitrogen gas.

EXAMPLE 5

Surface immobilization of hirudin-spacer conjugates by thiol ethers

Sulfo-LC-SPDP-or N-acetyl-homocysteine-hirudin was attached to surfacesby formation of a thiol ether bond. In brief, an agarose gel bearingeither a long-chain iodoacetyl group (0.5 ml SULFOLINK GEL®(cross-linked agarose, Pierce) or a maleimide (SulfOSMCC:Sulfosuccinimidyl 4-(maleimidomethyl) cyclohexane-1-carboxylate) wasreacted with either of the above hirudin derivatives (1.43×10⁻⁶ M in0.05M Tris-HCl, 0.005M EDTA-Na, pH 8.5) for 1 hour at room temperature.The gel was washed with 0.05M Tris, 0.005M EDTA-Na, pH 8.5; incubatedwith 0.05M cysteine, 0.05M Tris, 0.005M EDTA-Na, pH 8.5 for 1 hour;washed with 1M NaCl; then equilibrated with physiological saline, pH7.2.

EXAMPLE 6

Binding of NHS-LC-Biotin to Hirudin-Tyr-NH₂

NHS-LC-biotin (Sulfosuccinimidyl-6-(biotinamido)hexanoate) was attachedto hirudin containing an aryl amine on tyrosine by the following method:To hirudin-Tyr-NH₂ (0.143×10⁻⁶ M) in 0.04M sodium acetate, pH 5.0, wasadded a ten-fold molar excess of NHS-LC-biotin and the solution agitatedfor 2 hours at room temperature. Excess LC-biotin was removed bydesalting on BIO-GEL® P-6DG.

EXAMPLE 7

Attachment of Hirudin-Spacer Coniuqates by Avidin-Biotin Complexes

Hirudin-Tyr-LC-biotin conjugates were bound to soluble avidin,avidin-coated polystyrene beads (FLUORICON particles, Baxter Healthcare)or avidin-coated silicone rubber tubing at a 1:1 molar ratio byincubating in 0.02M sodium phosphate pH 7.4 for 1 hour at roomtemperature.

EXAMPLE 8

Anti-thrombin activity of attached hirudin analogs

The thrombin inhibition activity of rHV2-Phe³ Gln³³ Tyr³⁵ Lys⁴⁷ Asp⁶³ :(H-Tyr), and its derivatives, including spacer molecules ranging from200 to 67,000 molecular weight, were determined by incubating them withhuman thrombin, then measuring the residual thrombin activity as theinitial velocity of amidolysis ofH-D-Phenylalanyl-L-pipecolyl-L-arginine-p-nitroanalide dihydrochloride(Kabi, S-2238), where zero thrombin activity was 100% inhibition.Aliquots containing 0.13-1.3 picomoles of hirudin as (a) the analog,H-Tyr, (b) the analog with an aryl amine on Tyr³⁵ : (H-Tyr-NH₂), (c) theanalog with LC-biotin attached to the tyrosyl amine: (H-Tyr-B), (d) thebiotinylated analog attached to soluble avidin: (H-Tyr-B-Av), (e) thebiotinylated analog attached to avidin-coated beads: (H-Tyr-B-Av-Beads),(f) the analog with N-acetyl-homocysteine attached to the tyrosyl amineand to SULFOLINK GEL® (H-Tyr-AH-SLG), and (g) the analog with LC-SPDPattached to the tyrosyl amine and to SULFOLINK GEL® (H-Tyr-SPDP-SLG);were reacted with 1.3 picomoles of human alpha-thrombin in 0.05MTris-HCl, pH 7.4, 0.1% bovine serum albumin, for 1 hour at 22° C.; thencentrifuged. Aliquots of the supernatants were diluted with the Tris-BSAbuffer and the reaction initiated by addition of S-2238. Reactionvelocities were monitored at 410 nanometers and used to determinethrombin activity. Moles of hirudin analog and its derivatives in eachassay were determined by radioiodinated hirudin analog tracer. Theanalog H-Tyr retained virtually all of its specific thrombin inhibitionactivity during insertion of an amine into the tyrosine ring, attachmentof LC-biotin spacer, and when bound through the spacer to soluble avidinor to avidin-coated beads. However, when the analog was attached to asurface via the shorter spacer molecules, diminished specific thrombininhibitory activity was observed (Table I).

                  TABLE I                                                         ______________________________________                                        Hirudin Analog   Thrombin Inhibition                                          Tyr)             (Specific Activity: *% of H--                                ______________________________________                                        H--Tyr           100                                                          H--Tyr--NH.sub.2 99                                                           H--Tyr--B        99                                                           H--Tyr--B--AV    98                                                           H--Tyr--B--Av--Beads                                                                           97                                                           H--Tyr--AH--SLG   6                                                           H--Tyr--SPDP--SLG                                                                              61                                                           ______________________________________                                    

EXAMPLE 9

Preparation and anti-thrombin activity of hirudin-Tyr-PEG adduct.

To provide a soluble hirudin of increased mass similar to thebiotinylated hirudin attached through a spacer to soluble avidin asdescribed in Examples 7 and 8, methoxypolyethylene glycol (5 kD) wasdirectly bound to hirudin through an aryl amine on tyrosine by reactinghirudin-Tyr-NH₂, 0.143×10⁻⁶ M in 0.04M sodium acetate, pH 5.0, with afifty-fold molar excess of methoxypolyethylene glycol-succinimidylsuccinate (MPEGSS) for 30 min at room temperature. The reaction wasstopped by addition of excess glycine at pH 7 to inactivate residualMPEGSS. SDS-PAGE analysis of the reaction products demonstrated completetransformation of hirudin-Tyr-NH₂ to its PEG adduct. The adduct retained90% of the thrombin inhibitory activity of the hirudin-Tyr-NH₂ startingmaterial when evaluated by the chromogenic assay described in Example 8.This illustrates that mass-increasing PEG derivatives can be directlyattached to rHV2-Phe³ Gln³³ Tyr³⁵ Lys⁴⁷ Asp⁶³ (SEQ ID NOS: 1 & 2)through an aryl amine in the phenolic ring of Tyr³⁵ without significantloss of biological activity.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 204 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..195                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATTACGTTCACAGACTGCACAGAATCGGGTCAAAATTTGTGCCTCTGC48                            IleThrPheThrAspCysThrGluSerGlyGlnAsnLeuCysLeuCys                              151015                                                                        GAGGGAAGCAATGTTTGCGGTAAAGGCAATAAGTGCATATTGGGTTCT96                            GluGlySerAsnValCysGlyLysGlyAsnLysCysIleLeuGlySer                              202530                                                                        CAAGGATACGGCAACCAATGTGTCACTGGCGAAGGTACACCGAAACCT144                           GlnGlyTyrGlyAsnGlnCysValThrGlyGluGlyThrProLysPro                              354045                                                                        GAAAGCCATAATAACGGCGATTTCGAAGAAATTCCAGAAGAAGACTTA192                           GluSerHisAsnAsnGlyAspPheGluGluIleProGluGluAspLeu                              505560                                                                        CAATGAAAAATG204                                                               Gln                                                                           65                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       IleThrPheThrAspCysThrGluSerGlyGlnAsnLeuCysLeuCys                              151015                                                                        GluGlySerAsnValCysGlyLysGlyAsnLysCysIleLeuGlySer                              202530                                                                        GlnGlyTyrGlyAsnGlnCysValThrGlyGluGlyThrProLysPro                              354045                                                                        GluSerHisAsnAsnGlyAspPheGluGluIleProGluGluAspLeu                              505560                                                                        Gln                                                                           65                                                                            __________________________________________________________________________

What is claimed is:
 1. An antithrombogenic molecule having the aminoacid sequence of (SEQ ID NO: 2).